Open Access

Original Article

Do triple test results predict risk for

neonatal hyperbilirubinemia?
Hale Goksever Celik1, Engin Celik2,
Gokhan Yildirim3, Merih Cetinkaya4
ABSTRACT
Objective: Neonatal jaundice is the most common condition that requires hospital admission and
outpatient follow-up after discharge in neonates. The values of more than 17 mg/dL in term infants
are accepted as neonatal significant hyperbilirubinemia. We aimed to define if there is any relationship
between second trimester serum markers and neonatal severe hyperbilirubinemia to protect the neonates
from its neurological damage.
Methods: Total 1372 pregnant women were enrolled who had done triple test between April 2014 and 2015
and then given birth at our hospital. Our primary outcome was neonatal significant hyperbilirubinemia.
Results: The mean age of our study population was 27.9±5.6. A total of 59 patients had babies with
neonatal hyperbilirubinemia after exclusion of Rh incompatibility. We detected that the presence of in
vitro pregnancy, maternal health problems or poor obstetric history had no effect on the risk for neonatal
hyperbilirubinemia. Neonatal hyperbilirubinemia was related with low E3 levels. The ratios of AFP/E3 and
hCG/E3 were the most helpful to predict the neonatal hyperbilirubinemia.
Conclusions: According to our results, low E3 levels in the triple test result can be helpful to predict the
development of the neonatal hyperbilirubinemia. However, this is a bit expensive and many developing
countries may not afford it.
KEYWORDS: Maternal screening, Neonatal hyperbilirubinemia, Triple test, Screening tests, Estriol.
doi: https://doi.org/10.12669/pjms.334.12420
How to cite this:
Celik HG, Celik E, Yildirim G, Cetinkaya M. Do triple test results predict risk for neonatal hyperbilirubinemia? Pak J Med Sci.
2017;33(4):979-983. doi: https://doi.org/10.12669/pjms.334.12420
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1. Dr. Hale Goksever Celik, MD. INTRODUCTION

Department of Obstetrics and Gynecology,
2. Dr. Engin Celik MD.
Neonatal jaundice, also called as neonatal
Department of Obstetrics and Gynecology,
Istanbul University School of Medicine, significant or severe hyperbilirubinemia, is the
Istanbul, Turkey. most common condition that requires hospital
3. Gokhan Yildirim, MD.
Assistant Professor,
admission and outpatient follow-up after
Department of Obstetrics and Gynecology, discharge in neonates. Neonatal physiologic
4. Merih Cetinkaya, jaundice can be a result of increased bilirubin
Assistant Professor,
Department of Neonatology,
production secondary to accelerated destruction
1,3,4: Kanuni Sultan Suleyman Training and Research Hospital, of erythrocytes or decreased excretory capacity
Istanbul, Turkey. secondary to low levels of ligandin which is a
Correspondence: binding protein for bilirubin in hepatocytes and low
Dr. Hale Goksever Celik, activity of bilirubin-conjugating enzyme uridine
Astera park houses, a blok, stage 11, diphosphoglucuronyltransferase (UDPGT).
House 47, Küçükçekmece,
Istanbul, Turkey. Although the level for definition varies according
E-mail: hgoksever@yahoo.com to gestational age, the values of more than 14
* Received for Publication: January 25, 2017 mg/dL in preterm infants and 17 mg/dL in
* Accepted for Publication: July 1, 2017 term infants are accepted as neonatal significant

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Hale Goksever Celik et al.
hyperbilirubinemia. Many researchers have tried
to find a predictor for this condition over the
years. We hypothesized that abnormal function
of fetal liver could help us the prediction of
neonatal severe hyperbilirubinemia. As mentioned
before, one of the pathophysiological mechanisms
underlying neonatal hyperbilirubinemia may be
related with abnormal functions in liver. For that
reason, estriol (E3) and alpha-fetoprotein (AFP),
second trimester serum markers, could be used
as helpful markers. Because estriol constitutes 60-
70% of the total estrogens during pregnancy if the
fetal adrenal and liver are functional and AFP is
secreted during pregnancy from the fetal liver in a
very high amount.1 We aimed to define if there is
any relationship between second trimester serum
markers and neonatal severe hyperbilirubinemia to
protect the neonates from its neurological damage.
METHODS
This study is a retrospective case-control study.
Total 1372 pregnant women were enrolled who
had done triple test between April 2014 and
April 2015 and then given birth at Kanuni Sultan
Suleyman Training and Research Hospital. Healthy
neonates with gestation ≥34 weeks were included.
Multiple pregnancies, newborns with major
congenital malformations or neural tube defect,
patients with Rh incompatibility were excluded.
All demographic and clinical characteristics
including age, weight, obstetric history, presence
of any comorbid conditions, in vitro fertilization
pregnancy, smoking, gestational week during triple
test and birth, birth weight, type of delivery, gender
of baby, serum levels of E3, AFP and total bilirubin
concentrations were obtained from written or
electronic records. Gestational ages were estimated
by ultrasonographic dating of the pregnancies. All
maternal serum markers had been studied with
solid-phase competitive enzyme immunoassay
method. All results were converted into multiples
of the median (MoM) for each of the analytes.
Our primary outcome was neonatal significant
hyperbilirubinemia indicated by a bilirubin
concentration of ≥17 mg/dL. Our study was
designed retrospectively and conducted according
to the Helsinki Declaration. There was not ethical
approval because we collected data of the patients
from the records and we did not documented
any personal information. Also in our hospital,
informed consent is taken from every patient about
that medical information may be used in scientific
publications.
Pak J Med Sci 2017 Vol. 33 No. 4
Statistical Analysis: Statistical analysis was
performed with the Statistical Package for the Social
Sciences (SPSS Inc; Chicago, IL, USA) statistics 22.0
version for Windows. Difference in mean values
and characteristics between groups were analyzed
with independent samples t test and chi-square test.
Means were presented with standard deviation
(SD). p<.05 was considered statistically significant.
Thresholds for the association of abnormal maternal
serum triple analytes with neonatal significant
hyperbilirubinemia for this study were determined
by initially using receiver operating characteristics
(ROC) curves to ascertain the optimal cut-off for
each analyte. When a significant cut-off value
was observed, the sensitivity, specifity, positive
likelihood ratio values were presented. While
evaluating the area under the curve, a 5% type-I
error level was used to accept a statistically
significant predictive value of the test variables.
RESULTS
Total 1372 women who had done triple test
between April 2014 and 2015 and given birth at
Kanuni Sultan Suleyman Training and Research
Hospital were included. The mean age of our study
population was 27.9±5.6, whereas the median age
was 28. According to median age, the majority
of women in the group aged 28-51. The mean
gestational week was 17.3±1.0 during triple test,
38.3±2.7 during birth (Table-I). Most patients were
multiparous giving birth by vaginal route and
had no Rh incompatibility. A total of 59 patients
had babies with neonatal hyperbilirubinemia after
exclusion of Rh incompatibility as an important
reason for neonatal hyperbilirubinemia (Table-II).
Table-I: Demographic and clinical
characteristics of patients.
Characteristics Mean ± SD
Age 27.9±5.6
Weight 65.7±12.8
Gravide 2.6±1.4
Parite 1.2±1.0
Gestational week on triple test 17.3±1.0
E 3 1.00±0.40
hCG 1.17±0.72
AFP 0.99±0.55
Gestational week on birth 38.3±2.7
Birth weight 3168.9±629.9
E3, estriol; hCG, human chorionic gonadotropin;
AFP, alpha-fetoprotein.
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 Triple test and neonatal hyperbilirubinemia

Table-II: Distribution of demographic Table-III: Comparison of characteristics of the patients

and clinical characteristics. according to presence of neonatal hyperbilirubinemia.
Characteristics No. (%) Neonatal hyperbilirubinemia
Characteristics
Age Absent Present p
≤28 676 (49.3) Age
>28 696 (50.7) ≤28 641 (48.8) 35 (59.3) NS
Parite >28 672 (51.2) 24 (40.7)
0 346 (25.2) Parite
≥1 1026 (74.8) 0 332 (25.3) 14 (23.7) NS
Smoking ≥1 981 (74.7) 45 (76.3)
Absent 1223 (89.1) Smoking
Present 149 (10.9) Absent 1169 (89) 54 (91.5) NS
Chronic health problems (DM, etc) Present 144 (11) 5 (8.5)
Absent 1351 (98.5) Chronic health problems
Present 21 (1.5) (DM, etc)
In vitro fertilization pregnancy Absent 1292 (98.4) 59 (100) NS
Present 21 (1.6) 0
Absent 1360 (99.1)
In vitro fertilization
Present 12 (0.9)
pregnancy
Poor obstetric history
Absent 1301 (99.1) 59 (100) NS
Absent 1363 (99.3)
Present 12 (0.9) 0
Present 9 (0.7) Poor obstetric history
Route of labor Absent 1304 (99.3) 59 (100) NS
Vaginal birth 788 (57.4) Present 9 (0.7) 0
Cesarean section 584 (42.6) Route of labor
Adverse obstetric result Vaginal birth 752 (57.3) 36 (61) NS
(preeclampsia, IUGR, GDM,etc) Cesarean section 561 (42.7) 23 (39)
Absent 1259 (91.8) Adverse pregnancy
Present 113 (8.2) outcomes
Rh incompatibility Absent 1207 (91.9) 52 (88.1) NS
Absent 1362 (99.3) Present 106 (8.1) 7 (11.9)
Present 10 (0.7) DM, diabetes mellitus.
Neonatal hyperbilirubinemia
Absent 1313 (95.7) DISCUSSION
Present 59 (4.3)
Neonatal jaundice is the most common condition
DM, diabetes mellitus; IUGR, intrauterine growth that requires medical attention in neonates. Neonatal
restriction; GDM, gestational diabetes mellitus. physiologic jaundice can be a result of increased
bilirubin production secondary to accelerated
We detected that the presence of in vitro
destruction of erythrocytes or decreased excretory
pregnancy, maternal health problems or poor
capacity secondary to low levels of ligandin which
obstetric history had no effect on the risk
is a binding protein for bilirubin in hepatocytes
for neonatal hyperbilirubinemia. There are and low activity of bilirubin-conjugating enzyme
differences between patients with or without uridine diphosphoglucuronyltransferase (UDPGT).
neonatal hyperbilirubinemia based on clinical and Neonatal significant jaundice is defined as leveled
demographic characteristics although there is not of at 14 mg/dL at 4 days in preterm infants and
any statistical significance (Table-III). 17 mg/dL in term infants requiring phototherapy
In our study, neonatal hyperbilirubinemia was or exchange transfusion to lower the total serum
related with low E3 levels. If the MoM values were bilirubin concentration.2 Many factors including
compared with each other, the ratios of AFP/E3 race, geography, genetics, nutrition, maternal
and hCG/E3 were the most helpful to predict the factors such as drug usage, presence of diabetes
neonatal hyperbilirubinemia (Table-IV). mellitus, birth weight, gestational age and

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Hale Goksever Celik et al.

Table-IV: Relationship between neonatal hyperbilirubinemia and MoM values.

Cut-off value Area under curve Sensitivity (%) Specifity (%) Positive likelihood ratio
E3 0.435 0.45 98.3 2.5 1.01
0.865 52.5 40.6 0.88
hCG 1.045 0.51 52.5 50.1 1.05
1.115 50.8 55.2 1.13
AFP 0.845 0.48 54.2 44.3 0.97
0.875 50.8 47.6 0.97
AFP/E3 0.95 0.53 59.3 50.2 1.20
0.98 55.9 52.5 1.18
AFP/hCG 0.87 0.51 52.5 48.2 1.01
0.94 50.8 53.8 1.10
hCG/E3 1.10 0.53 59.3 50.6 1.20
1.11 57.6 51.6 1.19
E3, estriol; hCG, human chorionic gonadotropin; AFP, alpha-fetoprotein.

congenital infections can increase the incidence of
pathologic neonatal jaundice.
Pathologic neonatal jaundice is one of the leading
cause for hospital readmissions during the first
week of postnatal life especially in low-income
countries.3 This condition is important because
increased bilirubin concentrations may result in
deaths or various neurological impairments such as
intellectual deficits, epilepsy, sensorineural hearing
loss.4 The cause for these neurological deficits
is accumulation of bilirubin in brain, termed as
“kernicterus”. Early detection and management of
kernicterus prevents these consequences. The best
approach to prevent significant hyperbilirubinemia
and its consequences is bilirubin screening
before hospital discharge.5 But neonates with
uncomplicated delivery mostly are discharged 48
hours after birth, so the diagnosis of significant
hyperbilirubinemia may be delayed. Because of
that, outpatient follow-up is critical for detection
and management of kernicterus.
Many studies have been done to define an
early marker of neonatal jaundice for the early
detection of neonates at high risk of severe
hyperbilirubinemia to prevent long-term poor
results.6 Chou et al found a strong correlation
Table-V: Results of logistic regression analysis.
RR (95% CI) p
AFP 1.57 (1.17-2.09) 0.002
hCG 1.09 (0.87-1.38) NS
E3 0.74 (0.44-1.23) NS
AFP/E3 1.38 (1.17-1.64) <0.001
AFP/hCG 0.99 (0.90-1.08) NS
hCG/E3 1.21 (1.05-1.40) 0.009
E3, estriol; hCG, human chorionic gonadotropin;
AFP, alpha-fetoprotein; RR, risk ratio.
between cord blood hydrogen peroxide levels and
bilirubin concentrations.7 Alkaline phosphatase
level has been thought as a significant predictor
for the severe hyperbilirubinemia and bilirubin-
induced neurological damage.8 Bilirubin/albumin
ratio was thought as good a indicator of risk for
bilirubin neurotoxicity.9
Triple test is applied between 14 and 21
gestational weeks and determine the risk for
fetal chromosomal abnormalities combining
maternal serum levels of AFP, E3, human chorionic
gonadotropin (hCG) and maternal age although it
is not diagnostic.10 There is also another benefit of
triple test that many adverse pregnancy outcomes
such as preeclampsia may be understood with
these serum markers. The relationship between
neonatal severe hyperbilirubinemia and serum
levels of second trimester markers was investigated
in our study. Because we thought that the neonatal
hyperbilirubinemia which is an abnormality of the
liver function could be understood with the level of
serum E3, AFP, hCG and the ratio of these hormones
during second trimester.
Our results showed that neonatal significant
hyperbilirubinemia is associated with the low levels
of E3 with a high sensitivity and low specificity. The
absence of statistically significant relationship could
be explained as many factors influence the levels of
second trimester serum markers and the origin of E3
during pregnancy is primarily fetal adrenal rather
than fetal liver.
According to our results, low E3 levels in the triple
test result can be helpful to predict the development
of the neonatal hyperbilirubinemia. Being aware
the importance of early detection and management
of neonatal hyperbilirubinemia, further studies

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should be needed to find predictors for that
condition during pregnancy.
Declaration of interest: None.
Finding: None.
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Authors’ Contributions:
HGC study conception, protocol development,
editing the manuscript and preparing the first draft
of the article.
EC study conception, protocol development and
data extraction.
GY and MC study design, revised and edited the
manuscript.
All authors read and approved the final manuscript.
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